Interpretive Summary: Leafy vegetables such as lettuce are increasingly implicated in outbreaks of foodborne illness. However, pathogenic bacteria which are inside leaf tissues are not effectively removed by surface treatments such as chemical washes. Irradiation has been shown to inactivate leaf-internalized bacteria, but many aspects of targeting these protected pathogens remain unknown. Leaves of Iceberg, Boston, green leaf and red leaf lettuce were cut into pieces, submerged in a cocktail mixture of three isolates of the human pathogen Escherichia coli O157:H7 and subjected to a vacuum perfusion process to force internalize the bacterial cells into the intercellular spaces of the leaves. The inoculated leaves were then treated with a water wash, a wash with a sanitizing solution or various doses of ionizing radiation. The internalized bacteria were recovered from the leaves and the surviving bacterial populations counted. Water, and sanitizing solution washes each gave some reductions of the pathogen. In contrast, all doses of irradiation significantly reduced E. coli O157:H7 on all lettuce varieties examined with the efficacy of the greatest reduction obtained as 99.999% on Iceberg lettuce. This study has shown that irradiation is able to effectively reduce viable E. coli O157:H7 cells internalized in lettuce, and that the variety of lettuce influences the specific response. This information will be of value to processors of fresh cut leafy vegetables in designing effective irradiation protocols to reduce the risks associated with this commodity.

Technical Abstract:
Pathogenic bacteria internalized in leaf tissues are not effectively removed by surface treatments. Ionizing radiation has been shown to inactivate leaf-internalized bacteria, but many aspects of targeting these protected pathogens remain unknown. Leaves of Iceberg, Boston, green leaf and red leaf lettuce were cut into pieces, submerged in a cocktail mixture of three isolates of Escherichia coli O157:H7 and subjected to a vacuum perfusion process to force internalize the bacterial cells into the intercellular spaces of the leaves. The inoculated leaves were then treated with a 3 min water wash, a 3 min wash with a sodium hypochlorite sanitizing solution (300 ppm or 600 ppm), or various doses of ionizing radiation (0.25 - 1.5 kGy). Leaves were stomached to recover the internalized cells and survivors enumerated. Water, 300 ppm and 600 ppm washes each gave reductions of less than 1 log; these were statistically significant only in the case of green leaf lettuce. In contrast, all doses of irradiation significantly reduced E. coli O157:H7 on all lettuce varieties examined. The specific variety influenced the efficacy of irradiation. The greatest reduction obtained was 5 logs on Iceberg lettuce, treated with 1.5 kGy. The D10 values (the dose necessary to achieve a 1 log reduction) were significantly (P<.05) different among the varieties of lettuce tested, and ranged from 0.30 kGy (Iceberg) to 0.45 kGy (Boston). These values were observed to be notably higher than previously published irradiation D10 values for E. coli O157:H7 surface inoculated onto these four lettuce varieties. This study has shown that irradiation is able to effectively reduce viable E. coli O157:H7 cells internalized in lettuce, and that the variety of lettuce influences the specific response.